干扰素-γ诱导的自噬不足导致伴有鼻息肉的慢性鼻-鼻窦炎上皮细胞 p62 依赖性凋亡。
Interferon-γ-induced insufficient autophagy contributes to p62-dependent apoptosis of epithelial cells in chronic rhinosinusitis with nasal polyps.
机构信息
Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R., China.
出版信息
Allergy. 2017 Sep;72(9):1384-1397. doi: 10.1111/all.13153. Epub 2017 Apr 4.
BACKGROUND
Autophagy is a lysosomal degradation pathway that is essential for cell survival, differentiation, and homeostasis. This study aimed to investigate the contribution of autophagy to the pathogenesis of CRS with nasal polyps (CRSwNP).
METHODS
The expression of autophagic proteins [microtubule-associated protein 1 light chain 3B (LC3B)-II, autophagy-related proteins (Atg), and Beclin 1], substrate proteins (p62 and ubiquitinated proteins), and apoptotic signaling molecules [cysteine-aspartic protease-3 and cysteine-aspartic protease-8, and poly-ADP-ribose polymerase] in the sinonasal mucosa and nasal epithelial cells (NECs) was detected by immunohistochemistry and Western blotting. Autophagic vacuoles were observed with transmission electron microscopy. BEAS-2B cells and NECs were treated with rapamycin, bafilomycin A1, or various cytokines. In some experiments, cultured NECs were transfected with small interfering RNA targeting p62 (sip62) or Atg5 (siAtg5). Cultured cells were analyzed with Western blotting and flow cytometry.
RESULTS
Although autophagic protein expression and autophagic vacuole formation were increased in both eosinophilic and noneosinophilic CRSwNP, particularly in NECs, there was also an up-regulation of substrate proteins and apoptotic signaling molecules. IFN-γ, but not IL-4, IL-13, or IL-17A, simultaneously enhanced LC3B-II and p62 levels as well as cell apoptosis in BEAS-2B cells and/or normal NECs. Bafilomycin A1 up-regulated the levels of LC3B-II and p62 in polyp NECs and IFN-γ-treated normal NECs. IFN-γ-induced apoptosis of normal NECs was exaggerated by bafilomycin A1 and siAtg5. Sip62 suppressed apoptosis of polyp NECs and IFN-γ-treated NECs. IFN-γ protein levels were increased in both eosinophilic and noneosinophilic CRSwNP.
CONCLUSIONS
IFN-γ induces activated but insufficient autophagy and thus contributes to a degree to p62-dependent apoptosis of NECs in CRSwNP.
背景
自噬是一种溶酶体降解途径，对细胞存活、分化和内稳态至关重要。本研究旨在探讨自噬在伴有鼻息肉的慢性鼻-鼻窦炎（CRSwNP）发病机制中的作用。
方法
采用免疫组织化学和 Western blot 检测鼻黏膜和鼻上皮细胞（NEC）中自噬蛋白[微管相关蛋白 1 轻链 3B（LC3B）-II、自噬相关蛋白（Atg）和 Beclin 1]、底物蛋白（p62 和泛素化蛋白）和凋亡信号分子[半胱氨酸天冬氨酸蛋白酶-3 和半胱氨酸天冬氨酸蛋白酶-8 和多聚 ADP-核糖聚合酶]的表达。用透射电镜观察自噬空泡。用雷帕霉素、巴弗洛霉素 A1 或各种细胞因子处理 BEAS-2B 细胞和 NEC。在一些实验中，用靶向 p62（sip62）或 Atg5（siAtg5）的小干扰 RNA 转染培养的 NEC。用 Western blot 和流式细胞术分析培养细胞。
结果
尽管嗜酸性粒细胞和非嗜酸性粒细胞性 CRSwNP 中自噬蛋白表达和自噬空泡形成增加，尤其是在 NEC 中，但底物蛋白和凋亡信号分子也上调。IFN-γ而不是 IL-4、IL-13 或 IL-17A 同时增强了 BEAS-2B 细胞和/或正常 NEC 中 LC3B-II 和 p62 的水平以及细胞凋亡。巴弗洛霉素 A1 上调了息肉 NEC 和 IFN-γ 处理的正常 NEC 中 LC3B-II 和 p62 的水平。巴弗洛霉素 A1 和 siAtg5 加剧了 IFN-γ 诱导的正常 NEC 凋亡。sip62 抑制了息肉 NEC 和 IFN-γ 处理的 NEC 的凋亡。IFN-γ 蛋白水平在嗜酸性粒细胞和非嗜酸性粒细胞性 CRSwNP 中均增加。
结论
IFN-γ 诱导激活但不足的自噬，从而在一定程度上导致 CRSwNP 中 NEC 依赖 p62 的凋亡。
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